Our laboratory has focused on understanding the mechanisms controlling acellular Hbs oxidative and nitrosative toxicities in vitro and in vivo. Evidence is accumulating which suggests that reduction-oxidation redox reactions of free Hb and hemoglobin-based oxygen carriers (HBOCs), also known as blood substitutes do occur in vivo with some potentially serious clinical consequences. A primary focus of the research is based on the role of oxidants-mediated changes in acellular Hb under conditions that can mimic ischemia/reperfusion injury. Depending on whether host endogenous reductive mechanisms are employed or not, we will investigate Hb oxidative reactions in vivo using rats (ascorbate producing) or guinea pigs (ascorbate non-producing). The role of haptoglobin (Hp) and/or other antioxidant materials in supplementing endogenous antioxidant mechanisms will be investigated. For the investigation of effects of oxygen carrying HBOCs on cardiac performance under hypoxia, we will perfuse isolated hearts from rats with a number of Hbs and/or antioxidant materials. Previous experiences from our laboratory make us uniquely positioned to critically evaluate Hb oxidative processes with regard to heme iron, amino acids and protein structure both in vitro and in vivo and the implementation of any successful therapeutic strategies designed collectively within this program. Our specific aims for Core D are to: 1) Determine oxidative and nitrosative characteristics of acellular Hbs in the presence or absence of specific nanomaterials in vitro. Studies will include autoxidation rate determination, H2O2 induced heme iron oxidation, protein oxidative changes, heme degradation and nitric oxide (NO)-driven oxidation reactions as outlined in both project 2 and 3. 2) Determine redox transition of acellular Hbs in cell culture media and/or in isolated organ under normoxia and hypoxia as well as their ability to carry oxygen and whether any given candidate(s) will proceed to the next level of in vivo evaluation in an appropriate animal model. 3) Perform a 50-80% exchange transfusion of blood from guinea pigs or rats with individual acellular Hbs. HIF-1 a protein, and down stream genes such as erythropoietin (Epo), endothelial nitric oxide synthase (eNos), heme oxygenase (Ho-1) and vascular endothelial growth factor (Vegf) will be evaluated by conventional molecular biology techniques. This together with in vivo work carried out in the microcirculation in project 1, will allow us to determine what particular chemistry of a given Hb/or additive offers advantage over others in terms of oxygen carrying and redox stability of' the carrier molecule.